Oligonucleotides have been synthesized in vitro using organic synthesis methods. These methods include the phosphoramidite method described in Adams et al., J. Amer. Chem. Soc., 105:661 (1983), and Froehler et al., Tetrahedron Lett., 24:3171 (1983) and the phosphotriester method described in German Offenlegungsscript 264432. Organic synthesis methods using H-phosphonates are also described in Froehler et al., as well as in U.S. Pat. No. 5,264,566.
The phosphoramidite method of phosphodiester bond formation and oligonucleotide synthesis represents the current procedure of choice employed by most laboratories for the coupling of desired nucleotides without the use of a template. In this method, the coupling reaction is initiated by a nucleoside attached to a solid support. The 5'-hydroxyl group of the immobilized nucleoside is free for coupling with the second nucleoside of the chain to be assembled. Because the growing oligonucleotide chain has a 5'-hydroxyl available for reaction with a mononucleoside, the direction of synthesis is referred to as 3' to 5'.
Each successive mononucleoside to be added to the growing oligonucleotide chain contains a 3'-phosphoramidate moiety which reacts with the 5'-hydroxyl group of the support-bound nucleotide to form a 5' to 3' internucleotide phosphodiester bond. The 5'-hydroxyl group of the incoming mononucleoside is protected, usually by a trityl group, in order to prevent the uncontrolled polymerization of the nucleosides. After each incoming nucleoside is added, the protected 5'-hydroxyl group is deprotected, so that it is available for reaction with the next incoming nucleoside having a 3'-phosphoramidite group and a protected 5'-hydroxyl. This is followed by deprotection and addition of the next incoming nucleotide. Between each nucleoside addition step, unreacted chains which fail to participate in phosphodiester bond formation with the desired nucleoside are chemically "capped" to prevent their further elongation. This usually involves chemical acetylation.
A drawback to the phosphoramidite method, as well as to virtually all chemical methods of phosphodiester bond formation, is that the reaction must be performed in organic solvents and in the absence of water. Many of these organic solvents are toxic or otherwise hazardous. Another drawback to chemical synthesis is that each addition or cycle is at best 98 percent efficient. That is, following each nucleotide addition, at least 2 percent of the growing oligonucleotide chains are capped, resulting in a yield loss. The total yield loss for the nucleotide chain being synthesized thus increases with each nucleotide added to the sequence. For example, assuming a yield of 98 percent per nucleotide addition, the synthesis of a polynucleotide consisting of 70 mononucleotides would result in a yield loss of approximately 75 percent. Thus, the object nucleotide chain would have to be isolated from a reaction mixture of polynucleotides, 75 percent of which would consist of capped oligonucleotides ranging between 1 and 69 nucleotides in length.
Thus, a need exists for a method of synthesizing polynucleotides which improves the efficiency of phosphodiester bond formation and is capable of producing shorter chain oligonucleotides in higher yields and longer chain polynucleotides in acceptable yields. There is also a need for a polynucleotide synthesis system which is compatible with pre-existing polynucleotides, such as vector DNAs, so that a desired polynucleotide can be readily added onto the pre-existing molecules. The phophoramidite method is not compatible with "add-on" synthesis to pre-existing polynucleotides.
Enzyme catalyzed phosphodiester bond formation can be performed in an aqueous environment utilizing either single or double stranded oligo- or polynucleotides as reaction initiators. These reaction conditions also greatly reduce the use of toxic and/or hazardous materials. The 3' to 5' direction of synthesis inherent to the phosphoramidite method of phosphodiester bond formation, however, cannot be enzyme catalyzed. All known enzymes capable of catalyzing the formation of phosphodiester bonds do so in the 5' to 3' direction because the growing polynucleotide strand always projects a 3'-hydroxyl available for attachment of the next nucleoside. There are many classes of enzymes capable of catalyzing the formation of phosphodiester bonds. The polymerases are largely template dependent because they add a complementary nucleotide to the 3' hydroxyl of the growing strand of a double stranded polynucleotide. The template independent polymerases primarily catalyze the formation of single stranded nucleotide polymers. The ligases are template independent enzymes and form a phosphodiester bond between two polynucleotides or between a polynucleotide and a mononucleotide.
Use of a template independent polymerase requires protection of the 3'-hydroxyl of the mononucleotide in order to prevent multiple phosphodiester bond formations and hence repeated mononucleotide additions. This approach requires the synthesis of nucleotides which are not normally utilized by the polymerase. These modified nucleotides tend to adversely affect the reaction rate. The template independent approach is also hampered by the commercial availability of only a single template independent polymerase, namely terminal deoxynucleotidyl transferase (EC 2.7.7.31). Thus, there is a further need for a system which can utilize template dependent polymerases but does not require the use of modified nucleotides.
There are many available template dependent polymerases having various characteristics useful for de novo synthesis of polynucleotide chains. Template-dependent polymerases (TDPs) have traditionally been used for a variety of synthetic protocols that involve copying preexisting DNA or RNA strands. These protocols fall into five categories, -namely primer extension or PCR (e.g., PCR Technology: Principles and Applications for DNA Amplification, H. S. Erlich (ed.), IRL Press at Oxford University Press, Oxford, England (1989)); mutagenesis (e.g., Smith. M., "In vitro mutagenesis", Annu. Rev. Genet. 19:423-462 (1985)); Sanger-type sequencing techniques (e.g., H. G. Griffin, A. M. Griffin, DNA Sequencing Protocols: Methods in Molecular Biology, Vol. 23, The Humana Press, Totowa, N.J. (1993)); "filling-in" techniques (e.g., J. Sambrook, E. F. Fritsch, and T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)); and replacement synthesis (e.g., Sambrook et al., supra.). None of these categories, however, contemplate the use of a template-dependent polymerase for the creation of an original polynucleotide strand for which a complete complementary template strand is non-existent. Thus, an even further need exists for a method of synthesizing an object polynucleotide sequence which is not a strict complementary copy of any single pre-existing template, using a template dependent polymerase.
In sum, current gene synthesis technology is expensive, error-prone and very time consuming. The inaccuracy of these techniques is exacerbated because sequence errors are not usually detected until after the gene is cloned and expressed. At that time, correcting errors involves discarding all the work and starting over. Thus, having fast access to synthetic genes, whose sequences are assured, and at a reasonable price, would move de novo gene construction from a last resort to the method of choice for analysis of the function of gene products.